Presynaptic Mechanisms

• Evoked vs. spontaneous exocytosis• Quantal release—What is the evidence?• Alternative techniques for monitoring exocytosis• Calcium & exocytosis• Calcium channels• Molecular mechanisms underlying exocytosis• SNARE proteins• Synaptotagmin

Nerve-evoked and spontaneous synaptic

transmissionNote multiple release sites

Spontaneousminiature EPSPs

Nerve-evoked EPSP + AP

Synaptic transmission

Fatt & Katz, 1952

Quantal Hypothesis: Each evoked response is made up of a number of unitary events (quanta) that

are the size of spontaneous events. Probability determines just how many:

m = np

* *

*

*

m = mean quantal contentn = number of quanta (or release sites) availablep = probability of release

where

Some people consider “n” to be the number of release sites

Others consider “n” to be the total number of vesicles available for release

If only one vesicle can be releasedfrom any one release site in response to a single action potential (which seems to be the case for many synapses, most of the time), then these two ways of defining “n” will give the same value--so we won’t worry about resolving this now

“Suppose we have, at each nmj, a population of n units capable of responding to a nerve impulse.”

Del Castillo & Katz 1954

n = number of quantap = probability of release m = quantal content

“Suppose, further, that the average probability of responding is p…then the mean number of units responding to one impulse is m = np. Under

normal conditions, p may be assumed to be relatively large…However, as we reduce the Ca and increase the Mg concentration, the chances of responding are diminished and we observe mostly complete failures with an occasional

response of one or two units. Under these conditions, when p is very small, the number of units x which make up the epp in a large series of

observations should be distributed in the characteristic manner described by Poisson’s Law.” Del Castillo & Katz 1954

must be low

P(x) = probability that a particular # of quanta are releasedn = number of vesicles (or release sites) availablep = probability of releasem = mean quantal content

Where

must be large

(& each vesicle must behave identically and independently)

For the special case of failures (no release, x = 0)—which are easy to detect accurately—

this simplifies to:

e-mP(0) = m0

0!

1

1

Ln P(0) = - m

Substituting for P(0)=(Number of Failures)/(Number of Stimuli) and rearranging:

m = Ln (Total # of Stimuli/# of Failures)

m also is equal to Mean EPP amplitude

Mean mEPP amplitude* *spontaneous event aka single quantum

m calculated both ways can be compared for verification

For 1st, simply count failures

Then compare results from both methods in the same cell

del Castillo & Katz, 1954

For 2nd, compare mean amplitudes of spontaneous and evoked events

spontaneous

evoked

A = mean EPPB = mean mEPP

Quantal Hypothesis predicts:

ln C

/D

A/BA/B = loge C/D

C = # of stimuliD = # of failures

del Castillo & Katz, 1954

“Can the miniature discharges be attributed to molecular leakage of

transmitter from nerve endings?”

“If the miniature discharges were to be regarded as the local depolarizing effect of individual ACh molecules then… the application of ACh in solution should greatly increase the frequency of the miniature discharges; in fact, the steady depolarization which is produced by applied Ach would have to be regarded as a fusion of miniature e.p.p.'s…This conclusion, however, is contrary to our observations. A moderate concentration of ACh which depolarized the end-plates by a few mV, did not appreciably alter the frequency of the miniature discharges. The only noticeable change was a slight reduction of amplitudes...incompatible with thesuggestion that the miniature potential might be attributed to the action of one(or very few) ACh molecules.”

Fatt & Katz, 1952

or

+ ACh

?

EM Synapse

Are these vesicles the physical correlate of quanta?

Freeze-slam image of NMJ immediately after stimulation

“omega” shapes

Are they fusing vesicles?

Freeze-fracture EM of active zone

fused vesicles

bumps are some kind of integral membrane protein—calcium channels?

Recycling of synaptic vesicles

•Neurotransmitter release is quantal•Vesicles are quantal packets of transmitter

So far, all “real time” examples monitor exocytosis by measuring

a postsynaptic response using electrophysiology.

What techniques can be used to monitor presynaptically exocytosis

directly?

Capacitance measurements of vesicle cycling at a ribbon synapse

AMPEROMETRY uses electrochemistry to detect catecholamine release from chromaffin

cells, while CAPACITANCE measurements report changes in membrane surface area

amperometry

capacitance

Ales et al., 1999

Lipophilic fluorescent dyes can be used to monitor vesicle cycling at small CNS synapses

FM1-43 stains recycling vesicles

FM1-43 loaded NMJ destains with stimulation

Bill Betz video

FM1-43 fluorescent dye loaded vesicles

Murthy & Stevens, 1999

FM1-43 reveals quantal transmission in cultured hippocampal neurons when FM dye is loaded under

low Prelease conditions where only a few vesicles are expected to be released & take up dye

Under some conditions, release of transmitter can be observed without release of FM1-43 dye—this could indicate that release is occurring through a fusion pore that is big enough to let transmitter but not dye to get out What is a fusion pore?

Are there different modes of exocytosis—i.e. “kiss & run”?

Are there different modes of exocytosis—i.e. “kiss & run”?

FM dyes suggest “full fusion” of vesicles

Ryan et al 1996